We currently have a very poor understanding of why some infections by the Gram positive pathogen Streptococcus pyogenes result in mild self-limiting disease, while others progress to life-threatening tissue destructive diseases. To a large extent, this reflects our poor understanding of the critical events that occur during the initial stages of the interaction of the organism with human tissue. The long term goal of this project has been to characterize these events in molecular detail and to develop techniques to facilitate this analysis. A central observation has been that expression of a major fibronectin binding adhesin, protein F, is linked to pathways which sense oxidative stress. This suggests that S. pyogenes must adapt to the effects of toxic oxygen chemistry during the initial stages of infection and that sensing oxidative stress provides an important signal for expression of virulence. The testing of this hypothesis will be the focus of this proposal. Three specific aspects of this question will be addressed. Previous work has shown that prtF is regulated by several distinct pathways, one of which involves the transcriptional activator RofA. Both RofA-dependent and -independent pathways have a common and absolute requirement for a specific site in the prtF promoter that is bound by RofA. This suggests that other RofA-like proteins (RALPs) present in the genome may regulate prtF. Testing this will involve characterization of the DNA-recognition domain of RofA, and the contribution of RofA and RALPs to virulence and regulation of other genes whose promoters contain RofA-binding sequences. The second aspect will be directed at further characterization of the Gas locus, a two-component regulator o unusual structure. While Gas mutants grow normally under anaerobic conditions, they loose their ability to grow under aerobic conditions. Identification of the regulatory targets of this locus may reveal novel genes required for resistance to oxidative stress. The third component of the project will involve characterization of an induced resistance response to peroxide stress that involves no previously known peroxidase or regulator. Finally, the ability of these mutants to cause disease in a murine model of cutaneous disease will be evaluated. This will provide a direct test of the hypothesis that genes which are required for resistance to oxidative stress and growth in an aerobic environment will be important for survival in inflamed tissue. Since most of these genes are unique, a number of promising candidate drug targets may be identified.